Use of Extracts or Materials Extracted From Piper Cubeba L. as an Effective Component in a Drug for the Treatment of Cancer Diseases

ABSTRACT

Disclosed is a method for the preparation of a dry extract from the fruit of  Piper cubeba  L. In the disclosed method, in a first step, the fruit of  Piper cubeba  L. either is subjected to a steam distillation for the removal of essential oils and the distillate is removed or extracted at least once with a lipophilic phase and the lipophilic extract or extracts are removed; in a second step, the fruit, so treated, is extracted at least once either with at least one alcohol or with a mixture of at least one alcohol and water; and, in a third step, the extracted fruit parts are removed and an auxiliary material is added to the extract so obtained, which is then concentrated to a thick extract with an alcohol concentration of 0.1 to 10% (w/w), then dried and recovered.

The present invention is directed to the use of extracts or of extractcompounds from Piper cubeba L. as active components in a medicament forthe treatment of cancer.

Uses of Piper cubeba L. compositions are described for example inHunnius, Pharmazeutisches Wörter-buch, 8^(th) edition, 1998, pages 1084to 1085.

Herein are described popular applications, such as the treatment ofheadache as well as the use as diuretic, urine disinfectant andstomachic,

As drug is used the immature fruit, from which also the cubeben-oil, theessential oil of the cubeben, is obtained by steam distillation.

Oleum Cubebae is applied in the same indica-tions as the fruits.

According to J. Seidemann in “World Spice Plants”, Springer-Verlag,2005, page 291 Piper cubeba L. is used for the aromatization of liqueur,ginger bread, and honey bread. As product is used the essential oil thatis obtainable from the immature fruit.

In JP 2000-095649 A are described extracts, among others also from thecubeben fruit that are obtained by means of a hydrophilic solvent, forexample acetone, methanol and ethanol, or their mixtures with water.Thus, such extracts contain both the essential oil and hydrophilicsubstances.

These extracts shall act as testosteron-5α-reductase inhibitors. Therebythese extracts shall influence positively the growth of hair.

These extracts shall also serve for the treatment of benign prostatehyperplasia.

In this document are given in table 1 IC₅₀-values that are referred tothe inhibition of the testosteron-5α-reductase.

The extract of cubeben inhibits according to this table the enzyme for50% at a concentration of 0.79 mg/ml.

A. Chatterjee et. al., Jour. Indian Chem. Soc., Vol. 45, No. 8, 1968,pages 723 to 725 describes the spectral characteristics of the puresubstance Cubebin. This chemical compound was isolated from an alcoholicextract of the defatted fruits of Piper cubeba L.

A. Dasgupta et. al., Quart. J. Crude Drug Ret., 1.8, (1980), No. 1,pages 17 to 25 describes the use of the essential oil of Piper cubeba L.for the treatment of for example cystitis, and gonorrhoea.

Eun-Mi Choi et. al., Journal of Ethnopharmacology, Elsevier ScientificPublishers Ltd., 89, 2003, pages 171 to 175 describes anti-inflammatorycharacteristics of an extract prepared with 80% methanol from driedfruits of Piper cubeba L.

During a screening-process were tested several tropical medicinal plantsupon their activity against tumor cells in vitro.

It was found quite surprisingly that an ethanolic extract from immaturefruits of cubeben kills all tested tumor cells.

As predominantly the essential oil of the cubeben fruits is used inmedicine it was obvious to extract the fruits with a suitable extractingagent in order to obtain the essential oil.

This was realized by an exhausting extraction with hexane.

The fruits, exempted in this way from the essential oil, were for thesake of completeness extracted additionally with 90% aqueous ethanol inorder to obtain medium polar extract compounds.

Both the obtained essential oil and the ethanolic secondary extract werethen tested upon their activity against tumor cells.

As expected it was found that the obtained essential oil killed alltested tumor cells. This points to the fact that the observed cytotoxiceffect is of un-specific nature and thus demonstrates no antitumoreffect.

But quite surprisingly it was found that the ethanolic secondary extractindeed did not kill directly any of the tested tumor cells but changedin some tumor cells their proliferation behaviour.

Such tumor cells proved to be as especially sensitive against theethanolic secondary extract that need for their growth sex hormones asgrowth factors. As examples are mentioned the breast cancer cell lineMCF 7 and the prostate cancer cell line LnCAP. This observa-tion allowsthe conclusion that the proliferation inhibiting activity may not relyprimary on an inhibition of the testosteron-5α-reductase as this isirrelevant for the growth of the breast cancer cell line MCF 7.

It is an object of the present, invention to provide a process for thepreparation of an extract from fruits of Piper cubeba L.

This extract shall be free or nearly free from cytotoxic essential oils.

This extract shall inhibit the growth especially of such tumor cellsthat need for their growth sex hormones as growth factors.

This extract shall show anti-androgenic and/or anti-estrogenicactivities.

This extract shall antagonize the activities of the sex hormonedihydrotestosterone, abbreviated with DHT, especially its proliferationenhancing and anti-apoptotic effect on prostate cancer cells,

These objects are attained with the present invention.

The invention is characterized by the characteristics as defined in theindependent claims.

Preferred embodiments are defined in the dependent claims.

In the following part are described possible embodiments of the presentinvention.

Thereby is made also reference to the figures.

In the following part are described possible embodiments of the presentinvention.

Thereby is made also reference to the figures.

FIG. 1a shows the anti-proliferate effect of the extract preparedaccording to example 1 on LNCap and PC-3 cells.

FIG. 1b shows the anti-proliferate effect of the pure substance Cubebinon LNCap and PC-3 cells.

FIG. 2 shows the inhibition of the DNA-synthesis of LNCap cells with theextract prepared according to example 1.

FIG. 3 shows the anti-androgenic effect of the extract preparedaccording to example 1 on the androgen-dependent cell proliferation onLNCap cells.

FIG. 4 shows the anti-androgenic effect of the extract preparedaccording to example 1 on the DNA-synthesis of LNCap cells.

FIG. 5 shows the anti-estrogenic effect of the extract preparedaccording to example 1 on the DNA-synthesis of MCF-7 cells.

FIG. 6a shows the inhibiting effect of the extract prepared according toexample 1 and of the pure substance Cubebin on the activity of5α-reductase type II.

FIG. 6b shows the inhibiting effect of the known 5α-reductase inhibitor“Finasterid” on the activity of 5α-reductase type II.

FIG. 7a shows that TNF-α induces the apoptosis of the tumor cells independency of the dose, and that this effect is abolished completelywith DHT in the tumor cells.

FIG. 7b shows that the anti-apoptotic effect of DHT is abolished by theextract prepared according to example 1.

FIG. 8 shows that both the extract prepared according to example 1 andthe pure substance Cubebin inhibit the secretion of the prostatespecific antigen (PSA) in dependency of the respective dose.

FIG. 9 shows that the extract prepared according to example 1 inhibitsstrongly the with DHT induced secretion of the prostate specific antigen(PSA).

FIG. 10 shows that the androgen receptor concentration in LNCap cells isreduced increasingly in dependency of the dose both by the treatmentwith the extract prepared according to example 1 and by the treatmentwith the pure substance Cubebin.

The following examples illustrate the present invention.

Example 1 Preparation of a Liquid Extract

110 g of immature, dried fruits of Piper cubeba L. having a grindingfineness from 0.1 mm to 0.9 mm were extracted under stirring at atemperature between 10° C. and 20° C. during 8 hours with 0.5 liters ofhexane. The hexane layer charged with the essential oils and highlylipophilic components was then separated. This procedure was carried outonce more, whereby the extraction time was limited to 2 hours.

The so defatted fruits were then dried in a vacuum cabinet at atemperature of 40° C. until constancy of the weight. There were obtained92 g of defatted drug material.

Then the so treated fruits were extracted under stirring at atemperature between 20° C. and 30° C. during 2 hours with a mixture of90 parts by weight of ethanol and 10 parts by weight of water.

The weight ratio between the drug and the extraction mixture was 1:5.

The so extracted drug was separated by means of layer filtration. Therewere obtained 380 g of a dark brown liquid extract having a drysubstance content of 1.92 m/m %, corresponding to a yield of extractcompounds of 7.3 g absolute from 92 g defatted fruits.

This extract is denoted in the following part as P9605.

This extract contains 20 m/m % of Cubebin, referred to the dry substancecontent.

Example 2 Preparation of a Dry Extract

A liquid extract obtained according to example 1 was added dose by doseinto an evaporator at a temperature of 40° C., and the evaporation wasstarted under vacuum (300 mbar to 20 mbar) and elevated temperature (40°C. to 55° C.).

During the distillation the remaining part of the fluid extract wasadded continuously dose by dose into the evaporator until the totalamount of the fluid extract has been added and until in the obtainedspissum extract a dry substance content from 30 to 40 m/m % was reached.

There were obtained 20.0 g of spissum extract with a dark brown colour,free flowing and of homogenous consistency. The spissum extract showed adry substance content of 36.5 m/m 9%, what corresponds to a content ofextract compounds of 7.3 g.

This concentrated spissum extract was mixed homogenously with 7.8 g ofan aqueous 40 m/m % gum arabic solution and then dried in a dryer undervacuum at a pressure from 150 mbar to 10 mbar and a temperature from 40°C. to 55° C.

There were obtained 10.4 g of an ochre brown dry extract having acontent of 30 m/m % of gum arabic as auxiliary agent.

Example 3 Preparation of a Dry Extract-Oil Suspension

A liquid extract obtained according to example 1 was added dose by doseinto an evaporator at a temperature of 40° C., and the evaporation wasstarted under vacuum (300 mbar to 20 mbar) and elevated temperature (40°C. to 55° C.).

During the distillation the remaining part of the fluid extract wasadded continuously dose by dose into the evaporator until the totalamount of the fluid extract has been added and until in the obtainedspissum extract a dry substance content from 10 to 20 m/m % was reached.

There were obtained 54.0 g of spissum extract with a dark brown colour,free flowing and of homogenous consistency. The spissum extract showed adry substance content of 15.7 m/m %, what corresponds to a content ofextract compounds of 7.3 g.

This spissum extract of low viscosity was mixed with 6.8 g of middlechain triglycerides (Ph. Eur.) and 0.5 g of soya-lecithin (ÖAB 90) andadded dose by dose into an evaporator at a temperature of 40° C. Theevaporation of this mixture was carried out during such a long timeunder vacuum (300 mbar to 40 mbar) and at elevated temperature (40° C.to 50° C.) until in the obtained spissum extract a dry substance contentfrom 70 to 80 m/m % was reached.

There was obtained a viscous spissum extract that was then dried in adryer under vacuum at a pressure from 150 mbar to 10 mbar and atemperature from 40° C. to 55° C. until a dry substance content of 99.5m/m % was reached.

There were obtained 14.9 g of a dark brown dry extract-oil suspensionhaving a content of 49 m/m % of middle chain triglycerides and 3.36 m/m% soya-lecithin as auxiliary agents.

Example 4 Inhibition of the Cell Proliferation

With the liquid extract P9605 prepared according to example 1 werecarried out cell proliferation tests. As control was carried along atypical substance of content of the cubeben fruits, the lignan Cubebin.

For the measurement of the inhibition of the cell proliferation thisextract was added to LNCap and to PC-3 cells. The so treated cells werecultivated during 4 days in 10% FBS culture medium.

For comparison the pure lignan Cubebin, that is contained in the extractprepared according to the invention and according to example 1 in anamount up to 20 m/m % of the dry substance, was added also to LNCap andto PC-3 cells. The so treated cells were cultivated during 4 days in 10%FBS culture medium.

Thereby it was proceeded according to T. Lindl, Zell- und Gewebekultur,4^(th) revised edition, 2000, Spektrum Akademischer Verlag, Heidelberg.

All obtained datas are given in percents with regard to the solventcontrol (test without extract and without Cubebin); there are given theaverage values with Standard deviation from 4 experiments with 3-foldrepetition.

From the datas as shown in FIGS. 1a and 1b it is obvious that both theextract prepared according to the invention and the pure substanceCubebin show an anti-proliferate effect on LNCap and PC-3 cells independency of the respective dose.

The inhibition was more pronounced on LNCap cells than on PC-3 cells.

It is obvious from FIGS. 1a and 1b that the inhibitive activity of theextract P9605 prepared according to example 1 is much stronger than thiswould be explainable by its content of Cubebin. The extract containsonly 20 m/m % of Cubebin, but shows the same (LNCap) or a stronger(PC-3) inhibitive activity.

Example 5 Inhibition of the DNA Synthesis

With the liquid extract P9605 prepared according to example 1 werecarried out DNA synthesis tests.

For the measurement of the inhibition of the DNA synthesis the extractprepared according to the present invention was added to LNCap cells.The so treated cells were cultivated during 4 days in 10% FBS culturemedium.

Then was measured the amount of incorporated ³H-thymidin.

Thereby it was proceeded according to T. Lindl, Zell- und Gewebekultur,4^(th) revised edition, 2000, Spektrum Akademischer Verlag, Heidelberg.

All obtained datas are given in percents with regard to the solventcontrol (test without extract); there are given the average values withStandard deviation from 4 experiments with 3-fold repetition.

It is obvious from the datas shown in FIG. 2 that the extract preparedaccording to the present invention inhibits the DNA synthesis independency of the respective dose.

Example 6 Anti-Androgenic Effect on the Cell Proliferation

The anti-androgenic effect on the androgen dependent cell proliferationof the liquid extract P9605 prepared according to example 1 wasdetermined.

Thereby the extract prepared according to the present invention wasadded to LNCap cells. The so treated cells were cultivated during 6 daysin 10% CSS culture medium.

This cultivation was realized once without the addition ofdihydrotestosterone, abbreviated with DHT, and once with the addition of1 nM DHT.

Then was determined the influence of the extract prepared according tothe present invention on the cell proliferation of the tumor cells onthe basis of the DNA content.

All obtained datas are given in percents with regard to the solventcontrol (test without extract); there are given the average values withStandard deviation from 4 experiments with 3-fold repetition.

It is obvious from the datas shown in FIG. 3 that the extract preparedaccording to the present invention overexcites in dependency of the dosethe stimu-lating effect of DHT on the cell proliferation of the tumorcells and in addition lowers the basal proliferation of the cells.

It is known that DHT enhances the cell proliferation; see the controlvalue at zero.

Example 7 Anti-Androgenic Effect on the DNA Synthesis

The anti-androgenic effect on the DNA synthesis of the liquid extractP9605 prepared according to example 1 was determined.

Thereby the extract prepared according to the present invention wasadded to LNCap cells. The so treated cells were cultivated during 6 daysin 10% CSS culture medium.

This cultivation was realized once without the addition ofdihydrotestosterone, abbreviated with DHT, and once with the addition of1 nM DHT.

Then was measured the amount of incorporated ³H-thymidin.

Thereby it was proceeded according to T. Lindl, Zell- und Gewebekultur,4^(th) revised edition, 2000, Spektrum Akademischer Verlag, Heidelberg.

All obtained datas are given in percents with regard to the solventcontrol (test without extract); there are given the average values withStandard deviation from 4 experiments with 3-fold repetition.

It is obvious from the datas shown in FIG. 4 that the extract preparedaccording to the present invention overexcites in dependency of the dosethe stimu-lating effect of DHT on the DNA synthesis of the tumor cellsand in addition lowers the basal DNA synthesis of the cells.

It is known that DHT enhances the DNA synthesis; see the control valueat zero.

Example 8 Anti-Estrogenic Effect on the DNA Synthesis of Breast TumorCells

The anti-estrogenic effect on the DNA synthesis of breast tumor cells ofthe liquid extract P9605 prepared according to example 1 was determined.

Thereby MCF-7 cells were cultivated during 3 days in 10% CSS culturemedium to which were added different concentrations of estradiol.

This cultivation was realized once without the addition of the extractprepared according to the present invention and once with the additionof 10 μg/ml of the extract.

Then was measured the amount of incorporated ³H-thymidin.

Thereby it was proceeded according to T.

Lindl, Zell- und Gewebekultur, 4^(th) revised edition, 2000, SpektrumAkademischer Verlag, Heidelberg.

All obtained results are given in DPM (radio-active degradation perminute); there are given the average values with Standard deviation from4 experiments with 3-fold repetition.

It is obvious from the datas shown in FIG. 5 that the extract preparedaccording to the present invention stops completely or nearly completelythe stimu-lation of the DNA synthesis of breast cancer cells byestradiol.

It is known that estradiol enhances the DNA synthesis of breast cancercells; see the control value at zero.

Example 9 Inhibition of the 5α-Reductase Type II Activity

The inhibition of the 5α-reductase type II activity by means of theliquid extract P9605 prepared according to example 1 was determined.

The assay was realized with a homogenate of HEK293 cells that overexpress the 5α-reductase type II (Reichert W., Hartmann R. W. and JoseJ.; 2001, Journal Enzyme Inhibition, Vol. 16, 47-53).

The influence of the extract prepared according to the present inventionand of the pure substance Cubebin on the activity of the 5α-reductasetype II was determined by means of the measurement of the conversion of³H-testosterone in ³H-DHT.

As control substance was used the known 5α-reductase inhibitor“Finasterid”.

All obtained datas are given in percents with regard to the solventcontrol (test without extract); there are given the average values withStandard deviation from 4 experiments with 3-fold repetition.

It is obvious from the datas shown in FIG. 6a that both the extractprepared according to the present invention and the pure substanceCubebin show an inhibitive effect on the activity of the 5α-reductasetype II.

The inhibition is stronger with the extract than with the pure substanceCubebin.

The extract inhibits with an IC₅₀-value of 3.6 g/ml, whereas the puresubstance Cubebin inhibits with an IC₅₀-value of 9.9 μg/ml.

The course of the dose activity graph of the extract and of the puresubstance Cubebin are analogous to the course of the dose activity graphof the known 5α-reductase inhibitor “Finasterid” (FIG. 6b ).

Example 10 Increase of Apoptosis

The induction of apoptosis by means of the liquid extract P9605 preparedaccording to example 1 was determined.

As preliminary test was added for the measurement of the induction ofapoptosis the tumor necrosis factor TNF-α alone as well as incombination with 100 nM dihydrotestosterone, abbreviated with DHT, toLNCap cells. The so treated cells were cultivated during 2 days in 10%FBS culture medium.

The apoptosis of the cells was measured by ap-plying a commercialapoptosis-immuno-assay-kit in which are detected specifically the DNAand histon fragments that are present as mono and oligonucleosomes.

It is obvious from the datas shown in FIG. 7a that TNF-α induces theapoptosis of the tumor cells in dependency of the dose.

This effect is revoked completely or nearly completely with DHT in thetumor cells.

Analogous experiments were carried out with DHT alone as well as incombination with 10 μg/ml of the extract prepared according to thepresent invention.

It is obvious from the datas shown in FIG. 7b that the anti-apoptoticactivity of DHT is revoked by the extract prepared according to thepresent invention.

Example 11 Inhibition of the Secretion of the Prostate Specific Antigen

The inhibition of the prostate specific antigen (PSA) by means of theliquid extract P9605 prepared according to example 1 was determined.

Thereby in one experiment LNCap cells were cultivated during 2 days in10% CSS culture medium to which were added different concentrationseither of the extract prepared according to the present invention or ofthe pure substance Cubebin.

Then was measured the secreted PSA amount in the cell supernatant bymeans of an immuno-Assay. Additionally the amount of DNA was determined.

In FIG. 8 is shown the proportion in percents of the amount of PSA tothe amount of DNA.

There are given the average values with Standard deviation from 4experiments with 3-fold repetition.

It is obvious from the datas shown in FIG. 8 that both the extract andthe pure substance Cubebin inhibit the secretion of the prostatespecific antigen (PSA) in dependency of the respective dose.

In a second experiment LNCap cells were cultivated during 2 days in 10%CSS culture medium to which were added different concentrations ofdihydrotestosterone, abbreviated with DHT.

This cultivation was realized once without the addition of the extractprepared according to the present invention and once with the additionof 10 μg/ml of extract.

Then was measured the secreted PSA amount in the cell supernatant bymeans of an immuno-Assay. Additionally the amount of DNA was determined.

In FIG. 9 is shown the proportion in percents of the amount of PSA tothe amount of DNA.

There are given the average values with Standard deviation from 4experiments with 3-fold repetition.

It is obvious from the datas shown in FIG. 9 that the secretion of theprostate specific antigen (PSA) induced by DHT is strongly inhibited bythe extract prepared according to the present invention.

Example 12 Generation of Androgen Receptors

The influence of the generation of androgen receptors by means of theliquid extract P9605 prepared according to example 1 was determined.

Thereby in one experiment LNCap cells were cultivated during 2 days in10% FBS culture medium to which were added different concentrationseither of the extract prepared according to the present invention or ofthe pure substance Cubebin.

Then was determined the change of the amount of the androgen receptor bymeans of the Westernblot analysis.

In FIG. 10 are shown the bands of the androgen receptor.

The androgen receptor denseness in LNCap cells is reduced increasinglyin dependency of the dose both by the treatment with the extractprepared according to the present invention and by the treatment withthe pure substance Cubebin.

CONCLUSIONS

In the examples 1 to 3 is shown by which com-binations of process stepsextracts of cubeben fruits may be prepared that are free or nearly freeof essential oil, show new characteristics and met the objects of thepresent invention.

Examples 4 to 12 demonstrate the antitumor activity of the extractsprepared according to the present invention and illustrate the activitymechanisms that form the basis of the activity against hormone dependenttumor cells. These examples show the high therapeutical potential of theextracts prepared according to the present invention, especially for thetherapy of malign diseases which progression is influenced by female ormale sex hormones.

When considering the efficacy (IC₅₀: 3.6 μg/ml) of the extracts preparedaccording to the present invention against the human 5α-reductase incomparison to the activity mentioned in JP 2000-095649 A (IC₅₀: 790μg/ml) then it becomes obvious that the extracts prepared according tothe present invention have an about 200-fold higher activity and thusopen also for the treatment of the prostate hyperplasia quite newpossibilities.

1. A process for the preparation of a dry extract of fruits of Pipercubeba L. as active component in a medicament, characterized in that ina first step for the removal of the essential oils the fruits of Pipercubeba L. either are subjected to a steam distillation and thedistillate is removed, or are extracted at least once with a lipophilicphase and this lipophilic extract or these lipophilic extracts is (are)removed, in a second step the so treated fruits are extracted at leastonce either with at least one alcohol or with a mixture of at least onealcohol and water, and in a third step the extracted fruit parts areremoved, the so obtained extract after the addition of an auxiliaryagent is first concentrated to a concentration of alcohol between 0.1and 10 m/m % to a spissum extract, is then dried and obtained. 2-7.(canceled)
 8. The process according to claim 17, wherein the extractingdefatted fruit is conducted at a temperature from 20° C. to 60° C. andfor 2 to 4 hours. 9-10. (canceled)
 11. A method of treating cancercomprising the administration of an extract from Piper cubeba L., to apatient in need of such treatment.
 12. A method of treating at least onedisease, selected from the group consisting of prostate cancer,testicles cancer, breast cancer, uterus cancer, including theirmetastases, and benign prostate hyperplasia, comprising theadministration of an extract prepared according to the method of claim17 to a person in need of such treatment.
 13. A method for antagonizingthe activities of the sex hormone dihydrotestosterone comprising theadministration of an extract prepared according to the process of claim17 to a patient in need of such treatment.
 14. A pharmaceuticalcomposition for the treatment of a disease selected from the groupconsisting of cancer diseases, prostate cancer, testicles cancer, breastcancer, uterus cancer, including their metastases, and of benignprostate hyperplasia, comprising an extract prepared according to theprocess of claim
 17. 15-16. (canceled)
 17. A process for the preparationof a dry extract of fruit of Piper cubeba L. as active component in amedicament, comprising extracting defatted Piper cubeba L. fruitmaterial with at least one alcohol or with a mixture of at least onealcohol and water.
 18. A pharmaceutical composition comprising anextract prepared according to the process of claim 17 and a carrier.